Understanding the molecular wiring that controls the highly coordinated process of skeletal myogenesis in adult muscle repair and regeneration will have significant impact on human health issues ranging from muscular dystrophies to disease- and aging-related muscle atrophy. Previous work from our laboratory established the mammalian target of rapamycin (mTOR) signaling as a master regulator of myogenesis by controlling distinct stages of myogenic differentiation. In the current grant cycle we have uncovered new players and pathways, and brought into focus a unique myogenic mTOR signaling network. These discoveries have taken us into new directions of investigation with the promise of deeper molecular understanding of myogenic regulation. With a combination of biochemical, molecular, cellular and genetic approaches, and utilizing both in vitro and in vivo experimental systems, we aim to (1) dissect the Vps34 pathway upstream of mTOR and examine its function in muscle regeneration, (2) uncover the mechanism by which mTOR exerts a kinase-independent function in the regulation of IGF-II expression through the transcriptional regulator YY1 in myogenesis, and (3) study the mechanism and function of XPLN in negative regulation of mTOR and myogenesis. Our expertise in biochemical characterization of signal transduction mechanisms as well as in muscle biology, our strong preliminary data, and the unique animal models available or under development, put us in an ideal position to pursue the proposed studies. New regulators and novel molecular interactions in regenerative myogenesis will likely be uncovered, which could be explored in the future for therapeutic targets in muscular dystrophies and disease- and aging-related muscle atrophy.